Capping System

The S-adenosylmethionine (SAM) capping protocol is a method used to enzymatically cap RNA transcripts with a modified nucleotide, typically m7G (7-methylguanosine), at the 5' end.

This modification mimics the natural cap structure found in eukaryotic mRNA, which is important for mRNA stability, translation initiation, and nuclear export. Here's a general protocol for SAM capping:

Materials Needed:

• In vitro transcribed RNA
• S-adenosylmethionine (SAM)
• RNA capping enzyme (e.g., Vaccinia Capping Enzyme or T7 RNA Capping Kit)

Protocol:

1. Prepare Reaction Mix:

   • In a microcentrifuge tube, prepare a reaction mix containing:
     • In vitro transcribed RNA
     • S-adenosylmethionine (SAM)
     • Reaction buffer supplied with the RNA capping enzyme
     • Nuclease-free water to bring the reaction volume to the desired amount.

2. Incubate Reaction Mix:

   • Incubate the reaction mix at the appropriate temperature for the RNA capping enzyme. This is typically 37°C for Vaccinia Capping Enzyme or as specified by the manufacturer's instructions.

3. Terminate the Reaction:

   • After the incubation period, add an EDTA solution or heat the reaction mix to inactivate the RNA capping enzyme and stop the reaction.

4. Purify Capped RNA (Optional):

   • If necessary, purify the capped RNA from unincorporated SAM and other reaction components using methods such as RNA purification columns or phenol-chloroform extraction.

5. Assess Capping Efficiency:

• Analyze the capped RNA using methods such as capillary electrophoresis, agarose gel electrophoresis, or mass spectrometry to assess the efficiency of capping.

Notes:

• It's essential to use nuclease-free water, tubes, and buffers to avoid RNA degradation.
• Optimize the reaction conditions (e.g., SAM concentration, reaction time) for efficient capping of your RNA of interest.
• Capped RNA can be used in downstream applications such as in vitro translation, transfection, or structural studies.

This protocol provides a general overview of the SAM capping procedure.